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In Vitro Culture of Moss Bryum Coronatum Schwaegr.(Bryaceae) and It’S Phytochemical Analysis

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In Vitro Culture of Moss Bryum Coronatum Schwaegr.(Bryaceae) and It’S Phytochemical Analysis Innovare International Journal of Pharmacy and Pharmaceutical Sciences Academic Sciences ISSN- 0975-1491 Vol 6, Issue 9, 2014 Original Article IN VITRO CULTURE OF MOSS BRYUM CORONATUM SCHWAEGR.(BRYACEAE) AND IT’S PHYTOCHEMICAL ANALYSIS VIJAY KANT PANDEY1, RASHMI MISHRA1, RAMESH CHANDRA1* 1Department of Bio-Engineering, Birla Institute of Technology, Ranchi, India. Email: [email protected] Received: 25 Jul 2014 Revised and Accepted: 26 Aug 2014 ABSTRACT Objective: The purpose of the present investigation was to establish in vitro conditions for moss Bryum coronatum Schwaegr. And to carry out preliminary phytochemical screening of B. coronatum leaf extracts in different solvents. Methods: Fresh unopened, mature capsules were used as explant and surface sterilization of spores bearing capsule with different concentration of sodium hypochlorite (0.5, 1, 2, 4 and 8%) with different time duration. The Murashige and Skoog (MS) medium that contains different concentration(full,1/2, 1/4th, 1/8th strength) with different concentration of sucrose were used for culture this moss. Phytochemical screening were carried out using ethanol, methanol and ethyl acetate leaf extract of B. coronatum to identify various constitutes using the standard procedures. Results: Surface sterilization of spores of this moss was most effective in 4% commercial bleach for 1 min sterilization. The optimum condition for germination of spores and for proper growth of gametophytes B. coronatum on MS/4 medium strength with sucrose (1.5%), at pH 5.8 and temperature 22±2ºC with 16/8h: light/dark condition. Phytochemical analyses revealed the presence of alkaloids, terpenoids and saponins in all extracts. Conclusion: Four percentage NaOCl aqueous solutions are better for surface sterilization of moss sporophytes. MS/4 medium with 1.5% sucrose found the best medium for spore germination. Solvents extracts showed presence of alkaloids, terpenoids and saponins in all extracts. Keywords: In vitro, Bryum coronatum, Spores, Alkaloids, Terpenoids, Saponins. INTRODUCTION compound which included chlorophyll, proteins and common sugars and secondary compounds which have terpenoids, alkaloids and Bryophytes (liverworts, mosses and hornworts) are non-vascular phenolic compounds [17]. Terpenoids exhibit various important plants, with nearly 15,000 species worldwide of which pharmacological activities i. e., anti-inflammatory, anti-malarial, approximately 8,000 are mosses, 6,000 are liverworts, and are 200 anticancer, inhibition of cholesterol synthesis, anti-viral and anti- are hornworts [1], was probably reduced early during land plant bacterial activities [18]. Terpenoids are very important in attracting evolution[2,3,4]. Bryophytes possess the following main useful mites and consume the herbivorous insects [19]. characteristics: they have a very simple structure compare to higher plant. Dominant vegetative phase is haploid gametophytes and less Genetic engineering of terpenoid metabolism attracts bodyguards to chromosome number[5]. Ono et al (1988) reported that cell of Arabidopsis. Alkaloids are used as anaesthetic agents and are found bryophytes especially in suspension culture, as ideal materials for in various medicinal plants [20]. morphogenetic, genetic, biochemical, physiological and molecular studies. Bryophytes occur in nearly every ecosystem on earth and B. coronatum Schwaegr. belonging to family Bryaceae (Bryopsida) play a major role in the recycling of carbon and nutrients through which are densely tufted, yellowish-green plant. It stem is short; 5- growth and decomposition[6]. 10 mm long, often branched. Stem leaves imbricate, lanceolate to ovatelanceolate, Setae 1-1.5 cm long, reddish-brown. Its capsules are Nearly 40% of bryophytes are endangered at present and in urgent pendulous, oblong and reddish-brown, the apophysis slightly wider need of active protection and conservation. In nature, bryophytes than the urn and bulging, rounded-obtuse at base, warty when dry. propagated by means of asexual (by multiplication of gametophytes This moss is widespread in tropical to warm-temperate regions Asia, parts) and sexual (through spore) reproduction. Tissue culture Africa, North America, South America, Australia, and Oceanic islands. methods of mosses offer an alternative mean of vegetative It habitat on soil, bricks, cemented bricks and rocks. propagation. The pioneer example for a bryophyte especially moss The aim of this paper is to report establishment of axenic culture of developed into a scientific model organism was moss Physcomitrella moss B. coronatum and to search for the conditions for full plant patens Hedw. [7,8,9,10,11]. The main advantage of P. patens is the development. Such knowledge is essential for designing ex situ easy way of its axenic in vitro cultivation on solid mineral conservation schedules, for future introduction and reintroduction medium[12], in liquid culture in flasks and in bioreactor[13-14]. Unlike and for evaluationof the biotechnological potential. For development other higher plant, in vitro culture of mosses cannot be initiated by using of protocol for the full gametophyte of B. coronatum. Our data will leaf explants since surface sterilization causes cell death. contribute future conservation biology of this moss as well as other Phytochemicals are naturally occurring in plants, leaves and other related mosses. Preliminary phytochemical screening of B. vegetative parts and roots. These phytochemicals have a role in coronatum leaf extracts in different solvents like ethanol, methanol defense mechanism of plants and in protection of plants from and ethyl acetate is being reported. various diseases. Chemical constituents and ethno-bryology of MATERIALS AND METHODS bryophytes are not elaborated very well [15-16]. Bryophytes are extremely rich in phenols (flavonoids and bibenzyl derivatives), Fully developed plant of B. coronatum with sporophytes and terpenoids, glycosides, fatty acids and also some aromatic gametophyte were collected from BIT, Mesra campus, Ranchi, India compounds. It also considered as a remarkable reservoir of new, in spring of 2013. Fresh unopened, mature capsules were used as natural products or secondary compounds, many of which have the starting plant material. Collected plant materials were identified shown interesting biological activities. Phytochemicals are primary by Botanical survey of India (BSI), Kolkata, India. For this voucher Pandey et al. Int J Pharm Pharm Sci, Vol 6, Issue 9, 307-311 specimen were deposited as plant herbaria in BSI, Kolkata and also well in ice. Sulphuric acid was then added carefully. A colour change in department of Bioengineering, BIT Mesra. Collected plants with from violet to blue to green indicates the presence of a steroidal healthy, immature sporophytes that contain spores wash them nucleus (that is, a glycone portion of glycoside). thoroughly in running tap water for 5 min. Immature capsules of B. coronatum with operculum or both with operculum and calyptra, Tests for steroids and undamaged sporophytes were separated from the i. A red colour produced in the lower chloroform layer when 2 ml of gametophytes and washed in distilled water. In vitro cultures of B. or ethanol, methanol and ethyl acetate leaf extracts of moss was coronatum were initiated by surface sterilization of spores bearing dissolved in 2 ml of chloroform and 2 ml concentrated sulphuric acid capsule with different concentration of sodium hypochlorite (0.5, 1, was added in it, indicates the presence of steroids. 2, 4 and 8%) with different time duration. Then, rinsed them 3 times in cold, sterile distilled water for 5 min. Capsules were opened with ii. Development of a greenish colour when 2 ml of the organic extract a sterile needle and transferred onto solid medium. Capsules were was dissolved in 2 ml of chloroform and treated with sulphuric and opened aseptically under the laminar flow cabinet and spores were acetic acid indicates the presence of steroids. transferred with a sterile needle to petri dishes containing 20 ml solid medium (MS medium with different concentrations such as RESULTS AND DISCUSSIONS 1/2, 1/4th, 1/8th and its normal strength with and without sucrose. In vitro culture of profits usually started from sporophytes via spore The basal medium (BM) contained MS(Murashige and Skoog)[21] germination [22]. However in some species like Rhodobryum,spores mineral salts and vitamins, 100 mg/L myo-inositol, 0.70% (w/v) are not regularly available and also some species of bryophytes do agar (Bacteriological) without any supplements of growth not produce sporophytes regularly. For the establishment of axenic regulators. The medium pH was adjusted to 5.8 prior to autoclaving culture from gametophytes and sporophytes of bryophytes there is at 121ºC for 20 min. Cultures were grown at 22±2ºC under white effective concentration of commercial bleach for surface sterilization fluorescent tubes at photon fluorescent rate of 2s and a which to kill the xenic organisms on the plants and not to harm the day/night regime of 16/8h. Moss plants were sub-cultured after 1 plants at the same time[22,23,24,25]. Surface sterilization of the month interval. 47 μmol/m sporophytes was more successful since we choose the almost mature but unopened capsules. The higher concentration of Preparation of plant extract commercial bleach harms the spores which cause decrease in spores The leaves of B. coronatum was removed from the plants and then germination. Gametophytes of this moss are very delicate because
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